Disodium 6-hydroxy-5-[(2-methoxy-4-sulphonato-m-tolyl)azo]naphthalene-2-sulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In-Vitro Gene Mutation:

Ames test:

In bacterial gene mutation study, the test chemical in water at doses 100-10000 µg/Plate were observed to be not mutagenic in Salmonella typhimurium strains TA100,TA1535,TA97 and TA98  with and without addition of S9 liver fractions from Aroclor induced hamsters. The same has been observed for rats as well.

Chromosomal Abberation test: The test chemical did not induce chromosome aberrations in the CHO cells and hence it is not likely to classify as a gene mutant in vitro.

In-Vivo Gene mutation:

Mammalian Erythrocyte Micronucleus test: In a Mammalian Erythrocyte Micronucleus Test, the frequency of MNPCEs in the positive control group was increased (p < 0.01) in comparison with that in the negative control group. Thus, the test chemical was considered to be not mutagenic in nature.

Transgenic Rodent Somatic and Germ Cell Gene Mutation Assay: In Transgenic Rodent Somatic and Germ Cell Gene Mutation Assay, the mutagenic nature of test chemical was evaluated. As the MFs of cII genes in liver and glandular stomach tissues from test chemical-treated animals were not significantly higher than those in respective negative control animals, it is clear that the test chemical has no mutagenic potential.

In vivo Alkaline comet assay: The test chemical did not induce DNA damage in alkaline comet assay in the liver and stomach tissue treated orally and hence it is not likely to classify as a gene mutant in vivo.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from publication

Qualifier:

according to guideline

Guideline:

other: As mentioned below

Principles of method if other than guideline:

Gene mutation toxicity study of the test chemical

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 97

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

with 10 % & 30 % HLI = induced male Syrian hamster liver S9 and 10% & 30 % RLI = induced male Sprague Dawley rat liver S9

Test concentrations with justification for top dose:

100-10000 µg/Plate

Vehicle / solvent:

Water

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

For strains tested with S9

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

For strains TA100 and TA1535 tested in the absence of S9

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

For strain TA97 tested in the absence of S9

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

For strain TA98 tested in the absence of S9

Details on test system and experimental conditions:

PREINCUBATION : In the standard protocol (preincubation) for conducting the Ames assay, a test tube containing a suspension of one strain of Salmonella typhimurium plus S9 mix or plain buffer without S9, is incubated for 20 minutes at 37º C with the test chemical.
Control cultures, with all the same ingredients except the test chemical, are also incubated. In addition, positive control cultures are prepared; these contain the particular bacterial tester strain under investigation, the various culture ingredients, and a known potent mutagen.
After 20 minutes, agar is added to the cultures and the contents of the tubes are thoroughly mixed and poured onto the surface of Petri dishes containing standard bacterial culture medium. The plates are incubated, and bacterial colonies that do not require an excess of supplemental histidine appear and grow.
These colonies are comprised of bacteria that have undergone reverse mutation to restore function of the histidine-manufacturing gene. The number of colonies is usually counted after 2 days.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: No mutagenic potential observed

 Strain: TA100

Dose	No Activation  (Negative)		No Activation  (Negative)		30% RLI  (Negative)		30% HLI  (Negative)		10% RLI  (Negative)		10% HLI  (Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation
ug/Plate	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM
0	111	9.5	123	2.7	126	2.3	120	5	138	4.7	116	6.4
100	118	16.9	134	6.3	138	7.3	119	9.1	126	3.7	128	1.5
333	123	7.8	120	5	136	3.3	120	5.8	134	5.3	134	11.3
1000	105	4.6	123	6.1	132	5.7	131	2.9	122	10.5	136	4.9
3333	127	3.8	133	4.2	126	3.2	137	4.7	125	3.5	119	5.5
10000	122	5.3	110	5.5	116	8	116	6	136	10.2	126	8.5
Positive Control	845	23.7	861	20.1	483	10.7	544	17.6	548	16.5	592	7.9

 

Strain: TA1535

Dose	No Activation  (Negative)		No Activation  (Negative)		30% RLI  (Negative)		30% HLI  (Negative)		10% RLI  (Negative)		10% HLI  (Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation
ug/Plate	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM
0	10	1	12	1.5	11	1.2	12	1.2	14	1.9	13	1.5
100	10	.3	10	.9	10	0	11	.7	10	.9	9	.7
333	10	1.2	9	.6	10	.7	11	.9	13	3	8	.7
1000	10	.3	10	2.1	11	2.3	9	.3	13	1.3	11	1.3
3333	9	.3	9	.6	9	.9	12	1.2	12	2	10	1.2
10000	9	.3	10	.6	10	1	11	2.5	10	1	11	2
Positive Control	890	19.6	857	11.9	90	6.7	94	5.4	117	7.5	142	4.9

Strain: TA97

Dose	No Activation  (Negative)		No Activation  (Negative)		30% RLI  (Negative)		30% HLI  (Negative)		10% RLI  (Negative)		10% HLI  (Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation
ug/Plate	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM
0	140	6.5	163	9.7	165	8.6	167	15.1	189	11.1	178	3.2
100	148	11	161	8.8	164	3.2	175	7.2	194	3.5	191	3.2
333	140	2.5	158	14.7	170	10.8	164	3	184	8.5	163	3.7
1000	154	3.8	167	5.5	164	17.7	176	6.4	178	5.2	165	8.5
3333	131	4.2	138	6.1	165	12.3	164	8.7	199	.3	180	16
10000	120	3.8	163	21.8	171	2.1	156	15.6	192	9.5	183	20.7
Positive Control	426	16.4	458	5.6	510	7	631	24.1	541	13.5	565	22.6

Conclusions:

In bacterial gene mutation study, the test chemical in water at doses 100-10000 µg/Plate were observed to be not mutagenic in Salmonella typhimurium strains TA100,TA1535,TA97 and TA98 with and without addition of S9 liver fractions from Aroclor induced hamsters. The same has been observed for rats as well.

Executive summary:

Genetic toxicity in vitro study was assessed for test chemical. For this purpose AMES test was performed.The test material was exposed to Salmonella typhimurium TA100, TA1535, TA98 and TA97 in the presence and absence of metabolic activation S9. The concentration of test material used in the presence and absence of metabolic activation were 0, 10, 30,100, 333, 1000 and 3333 µg/plate. Mutagenic effects were observed in neither strains, in the presence and absence of metabolic activation. Therefore test chemical was considered not to be mutagenic in Salmonella typhimurium TA100, TA1535, TA98 and TA97 by AMES test. Hence the substance cannot be classified as gene mutant in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from publication

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

Determination of genotoxicity of the test chemical by Mammalian Erythrocyte Micronucleus test.

GLP compliance:

no

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

other: CD1 mice (Crlj:CD1)

Details on species / strain selection:

No data

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Japan
- Age at study initiation: 6 weeks
- Weight at study initiation: No data
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study: No data
- Housing:No data
- Diet (e.g. ad libitum): CRF-1 pellet feed, Oriental Yeast
- Water (e.g. ad libitum): ad libitum
- Acclimation period:7 days before treatement

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data
- Humidity (%): No data
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): No data

IN-LIFE DATES: From: To:No data

Route of administration:

oral: gavage

Vehicle:

Vehicle(s)/solvent(s) used: 0.5% carboxymethylcellulose sodium
- Justification for choice of solvent/vehicle: The test chemical was soluble in 0.5% carboxymethylcellulose sodium

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:The test chemical was dissolved in 0.5% carboxymethylcellulose sodium solution prior to administration.

DIET PREPARATION
- Rate of preparation of diet (frequency):No data
- Mixing appropriate amounts with (Type of food): No data
- Storage temperature of food:No data

Duration of treatment / exposure:

2 days

Frequency of treatment:

Daily with a 24 hr interval

Post exposure period:

No data

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

30 mice
6 mice per group

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin C

Tissues and cell types examined:

Bone marrow cells

Details of tissue and slide preparation:

Tissue preparation:
Mice were sacrificed by cervical dislocation at 23-24 h after the final treatment.Femurs was removed and bone marrow cells were flushed from femurs into centrifuge tubes using calf serum. Cells were then resuspended in Dulbecco's phosphate-buffered saline and were centrifuged, and supernatants were then removed. These procedures were performed twice, and then cells were fixed in 10% neutral-buffered formalin solution. After exchanging the fixative twice by centrifugation, cells were re-suspended in formalin solution, and the cell suspension was filtered through a cell strainer.

Slide preparation:
Fixed cell suspensions were dropped onto cover slips and were immediately placed on acridine orange coated slides. Two thousand polychromatic erythrocytes (PCEs) per animal were analyzed using a fluorescent microscope with a 1000 lens, equipped with a blue excitation filter and a barrier filter, and numbers of micronucleated polychromatic erythrocytes (MNPCEs) were counted. To investigate the influence of the test substance on bone marrow cell proliferation, numbers of PCEs in a total of 500 erythrocytes were counted.

Evaluation criteria:

No data

Statistics:

Frequencies of MNPCEs in treatment and positive control groups were compared with those in the negative control group using conditional binomial tests.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Remarks:

0.5% carboxymethylcellulose sodium

Negative controls validity:

valid

Remarks:

0.5% CMC

Positive controls validity:

valid

Remarks:

Mitomycin C

Remarks on result:

other: No mortality observed

Additional information on results:

In micronucleus tests, no significant differences in MNPCEs were found between test chemical treatment groups and the negative control group. The frequency of PCEs, which offers an index of the influence of the test substance on bone marrow cells, did not differ between any of the treatment groups and the negative control group.The frequency of MNPCEs in the positive control group was increased (p ≤ 0.01) in comparison with that in the negative control group. No detahs, clinical signs and body weight changes were observed in control and tratment groups.

Table: The results of micronucleus test in bone marrow of CD1 mice after test chemical treatment.

Compound	Dose(mg/kg)	No. of animal	% MNPCE^a	% PCE
Control	0	5	0.14±0.07	63.3±8.1
Test chemical	500	5	0.20±0.04	61.8±5.1
	1000	5	0.14±0.07	61.4±9.1
	2000	5	0.17±0.14	62.9±5.0
MMC	1	5	3.66±0.94**	56.0±8.1

**:p < 0.01, significant difference from control (Kastenbaum and Bowman method, upper-trailed).

Control: negative control (0.5% Carboxymethylcellulose sodium, 10 ml/kg).

MMC: positive control (Mitomycin C, dose once a day, for 2 days, i.p., 1 days after administration).

^aPolychromatic erythrocytes possessing one or more than one micronuclei (MNPCEs) were counted.

Conclusions:

In a Mammalian Erythrocyte Micronucleus Test, the frequency of MNPCEs in the positive control group was increased (p ≤ 0.01) in comparison with that in the negative control group. Thus, the test chemical was considered to be not mutagenic in nature.

Executive summary:

In a Mammalian Erythrocyte Micronucleus Test, the mutagenic nature of test chemical was evaluated in male CD1 mice (Crlj: CD1) mouse. The mice were dosed orally by gavage at a concentration of 500, 1000 and 2000 mg/kg bw. 0.5% carboxymethylcellulose sodium was used a vehicle and Mitomycin C was used as a positive control substance. 30 mice (6 mice /group) were dosed daily at an interval of 24 hrs for 2 days. Bone marrow cells were examined. The target tissues were treated to achieve cells, slide preparation of the cells was done and later the slide was used for electrophoresis. Frequencies of micro nucleated polychromatic erythrocytes (MNPCEs) in treatment and positive control groups were compared with those in the negative control group using conditional binomial tests.In micronucleus tests, no significant differences in MNPCEs were found between test chemical treatment groups and the negative control group. The frequency of PCEs, which offers an index of the influence of the test substance on bone marrow cells, did not differ between any of the treatment groups and the negative control group. The frequency of MNPCEs in the positive control group was increased (p ≤ 0.01) in comparison with that in the negative control group. No deaths, clinical signs and body weight changes were observed in control and treatment groups. Thus the test chemical was considered to be not mutagenic in nature.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In-Vitro Gene mutation studies:

Ames test:

Data available for the test chemical was reviewed to determine the mutagenic nature of test chemical, The studies are as mentioned below:

1. Genetic toxicity in vitro study was assessed for test chemical. For this purpose AMES test was performed.The test material was exposed to Salmonella typhimurium TA100, TA1535, TA98 and TA97 in the presence and absence of metabolic activation S9. The concentration of test material used in the presence and absence of metabolic activation were 0, 10, 30,100, 333, 1000 and 3333 µg/plate. Mutagenic effects were observed in neither strains, in the presence and absence of metabolic activation. Therefore test chemical was considered not to be mutagenic in Salmonella typhimurium TA100, TA1535, TA98 and TA97 by AMES test. Hence the substance cannot be classified as gene mutant in vitro.

2. Ames assay was performed to determine the mutagenic nature of test chemical. The test chemical was studies for its mutagenic nature using Salmonella typhimuruim strains TA1537, TA1535, TA100 and TA98 with and without S9 metabolic activation system. The test chemical was mixed with DMSO and used at dose level of 10-250 mg/plate. 9-aminoacridine, 2aminoflourine and N-methyl-N-nitrosoguanidine served as a positive control with and without S9 metabollic activation. Mutagenicity was evaluated by counting histidine-revertant (his') colonies arising on these plates. As test chemical did not produce mutation in Salmonella strainTA98, TA100, TA1535, and TA 1537,Therefore it is considered to be negative for gene mutation in vitro.

3. Ames assay was performed to determine the mutagenic nature of test chemical. The test chemical was studies for its mutagenic nature using Salmonella typhimurium TA98, TA1535, TA1537 and Saccharomyces cerevisiae strain D4 with and without S9 metabolic activation system.Toxicity tests were conducted to identify the 12.5%, 25% and 50% killing doses. If no toxicity was found, a maximum dose of 5% was used as the highest dose concentration. The dose concentrations used were 0.625, 1.25, 2.5, 5.0% or 10, 100, 1000, or 5000 micrograms per plate. Positive controls used for experiment without S9 metabollic activation were ethylmethanesulfonate (EMS); methylnitrosoguanidine (MNNG); 2-nitrofluorene (NF); quinacrine mustard (QM) and postive controls with activation included 2-anthramine (ANTH); 2-acetylaminofluorene (AAF); 8-aminoquinoline (AMQ); dimethylnitrosamine (DMNA). The maximum dose of 5% was used, since 50% survival was not determined. Slight toxic effects noted at 5%. The test substance did not exhibit genotoxic activity with or without metabolic activation in the AMES assay using SAL TA98, TA1535 or TA1537 plate overlay method. Therefore the test chemical can be considered to be not mutagenic in nature.

4. Salmonella Mutagenicity Tests of test chemical was performed in Salmonella strainTA98, TA100 both in the presence and absence of S9 metabolic activation system. First, the test chemical was tested with UV irradiation. Aqueous solutions of test chemical were irradiated by a UV lamp. The concentration of the test chemical was 0.15 g/50 ml, with distilled water as solvent. The test chemical was constantly stirred and irradiated for 14 days. The degrees of decolouring were measured by visible light spectrophotometry. The λ max of test chemical was 525, 500, 500, 630 and 600 nm.

Again the test chemical was testd according to Ames assay were the test chemical was dissolved in distilled water (Solvent control) and the positive controls were AF-2 at the maximum concentration of 0.1 mg/plate for TA98 and TA100 in the absence of S-9 mix, and 2AA at the maximum concentration of 1.0 mg/plate for TA98 and TA100 in the presence of S-9 mix. The dose concentrations used were 0, 750, 1500 and 3000 µg/plate. Plates were observed for mutant colonies. The relationship between the number of His+ revertant colonies and the dose of test chemical was used for linear regression. The test chemical was observed to non-mutagenic before and after UV irradiation. In addition, the degree of decolouring of test chemical was small, indicating that the test chemical is stable under UV irradiation.As no mutagenic activity was observed before and after UV irradiation, the test chemical was considered to be not mutagenic in nature.

5. Salmonella Mutagenicity Tests of test chemical was performed in Salmonella strain TA98, TA100, TA1535, TA 1538 and TA 1537 both in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO to make dose concentration of 50,250 and 1000 µg/plate. The S-9 fractions of Aroclor 1254-induced, female Sprague-Dawley rat liver were prepared. The test was carried out according to pre incubation (paper disk) method. Positive controls used were 4-nitroquinoline-N-oxide (Salmonella typhimurium strain TA 1538 and TA 98), 9-aminoacridine (Salmonella typhimurium strain TA 1538), ethylmethanesulphonate (Salmonella typhimurium strain TA 100), methylmethanesulfonate (Salmonella typhimurium strain TA 1537), Anthragallol and 2-Anthramine for Salmonella typhimurium strain TA 98 with and without metabollic activation. Sodium dithionite was used as negative control. The number of revertants observed in test plates were in the decreasing order as compared to positive control. Therefore the test chemical was considered to be negative for gene mutation study.

Based on the observations made, the test chemical does not exibit gene mutation in vitro . Hence it is not likely to classify as a gene mutant in vitro as per the criteria mentioned in CLP regulation.

Chromosomal Abberation:

Data available for the test chemical was reviewed to determine the mutagenic nature of test chemical, The studies are as mentioned below:

1. In the study conducted, test chemical was examined for its ability to cause mutagenic changes when tested on Chinese hamster lung (CHL) or Chinese hamster ovary (CHO) fibroblast cells. Compund is represented by calculated structural descriptors that encode topological, geometric, electronic, and polar surface features. A genetic algorithm (GA) employing a k-nearest neighbor (kNN) fitness evaluator is used to iteratively search a reduced descriptor space to find small, information-rich subsets of descriptors that maximize the classification rates for clastogenic and nonclastogenic responses. To further improve modeling, a similarity measure using atom-pair descriptors is employed to create more homogeneous data subsets. Negative responses were obtained in all three different data sets are examined. Hence,based on the results of this study, the test substance was not considered to be mutagenic under the conditions of this test.

2. In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. The study was performed using CHO-WBL cells in the presence and absence of exogeneous metabolic activation system. The test chemical was dissolved in DMSO and used at dose levels upto 5 mg/mL. In the chromosome aberration assay without activation, cells were exposed to the test chemical for 8 hr. The test chemical was washed off, and the cells were treated with 0.1µg/ml Colcemid for 2-2.5 hr. With metabolic activation, the cells were exposed to the test chemical plus the metabolic activation mixture for 2 hr, washed, incubated for 8 hr, and then treated with Colcemid for 2-2.5 hr. A delayed harvest was used in the aberration assay in most instances when cell cycle delay was observed in the SCE assay. In these tests the cell growth period was extended to about 20 hr. Cells were harvested. Air-dried slides were coded and stained with Giemsa. One hundred to 200 cells from each of the three highest scorable doses were analyzed and the chromosomal aberrations were scored. The test chemical did not induce chromosomal aberrations when tested to toxicity. Precipitate was evident at doses of 250µg/ml and above. Based on the observations made, the test chemical did not induce chromosome aberrations in the CHO-WBL cells in the presence and absence of exogeneous metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

3. In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of test chemical. The study was performed using Chinese hamster ovary cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in serum free culture medium and used at dose level of 0, 1000, 1600, 3000 or 5000 µg/plate. Concurrent solvent and negative control chemicals were also included in the study. Approximately 24 hr prior to cell treatment, 1.2 x 10⁶cells were seeded per 75 cm2 flask. For assays without metabolic activation, the cells were treated for about 10 hr. Colcemid was added 2-3 hr prior to cell harvest by mitotic shake-off. In the test protocol for assays with metabolic activation cells were harvested approximately 11 hr after removal of the S9 fraction. Colcemid was added 2 hr prior to harvest. Slides were stained in 6% Giemsa for 5-10 min. One hundred cells were scored for each dose in early studies and 200 cells per dose in later studies. All slides except high-dose positive controls were coded. Only metaphase cells in which the chromosome number was between 19 and 23 were scored. The test chemical did not induce ABS at doses up to 5000µg/ml in either the presence or the absence of S9. Based on the observations made, the test chemical did not induce chromosome aberration in Chinese hamster ovary cells in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

4. The cytogenetic effects on cultured cells of the test chemical was evaluated using, Chinese hamster cultured cells (CHL / IU, hereinafter abbreviated as CHL ) to perform an in vitro chromosomal aberration test. In order to determine the concentration used for the chromosomal aberration test, the cell proliferation inhibition test was carried out, and as a result, the concentration showing growth inhibition of about 50% in the direct method was 0.37 mg / ml. On the other hand, in the metabolic activation method, no growth suppressive effect exceeding 50% was observed in any treated concentration group (0.16 to 5.00 mg / ml) in the presence or absence of S9 mix. Therefore, in the chromosome aberration test, the treatment concentrations of 0.37 mg / ml by the direct method and 5.0 mg / ml by the metabolic activation method were respectively set to high concentrations, and the half concentration thereof was defined as medium concentration, the concentration at ¼ was low Concentration.An aqueous solution of 0.5% sodium carboxymethyl cellulose (Nacalai Tesque, Lot No. M9G8053) was used as a solvent. The stock solution was suspended in a solvent to prepare a stock solution, and then the stock solution was serially diluted with a solvent to prepare a test substance preparation solution of a predetermined concentration. The test substance preparation solution was added so as to be 10% (v / v) of the culture solution in all tests. The positive controls used were mitomycin (for without S9) and Cyclophosphamide (for with S9). Two hours before the end of the culture, colcemid was added to the culture solution to a final concentration of about 0.1 μg / ml. Chromosome specimens were prepared according to a conventional method. Six slide specimens were prepared for each dish. The prepared specimens were stained with 3% Giemsa solution for about 10 minutes.Analysis of chromosomes was performed based on the classification method by the Japan Society of Environmental Mutagenesis, Mammalian Examination (MMS) Subcommittee 1), and the presence or absence of structural abnormality such as chromosome type or chromosome type gap, The presence or absence of cells (polyploid) was observed. For structural abnormalities, 200 metaphase cells per group and 800 division metastatic cells were analysed for multiplicative cells.By directly treating CHL cells for 24 h and 48 h, no chromosomal structural abnormalities or polyploid cell inducing effects were observed in all treatment groups. In addition, in any concentration group in the presence of S9mix in the metabolic activation method, the inducing action of chromosomal structural abnormality was not observed. In the low concentration group (1.3 mg / ml) in the absence of S9 mix, a significant increase in structural abnormality was observed, but the frequency was 2.5% and the judgment was negative. In the high concentration group (5.0 mg / ml) in the absence and presence of S9 mix, a significant increase in ploidy cells was observed, but the frequency of occurrence was less than 5% and the judgment was negative. From the above results, it was concluded that the test chemical does not induce chromosomal abnormalities in CHL cells in vitro under the above test conditions.

Based on the observations made, the test chemical does not induce chromosomal abnormalities in CHL cells in vitro. Hence it is not likely to classify as a gene mutant in vitro as per the criteria mentioned in CLP regulation.

In-Vivo Gene mutation studies:

Mammalian Erythrocyte Micronucleus test:

Data available for the test chemical was reviewed to determine the mutagenic nature of test chemical; the studies are as mentioned below:

In a Mammalian Erythrocyte Micronucleus Test, the mutagenic nature of test chemical was evaluated in male CD1 mice (Crlj: CD1) mouse. The mice were dosed orally by gavage at a concentration of 500, 1000 and 2000 mg/kg bw. 0.5% carboxymethylcellulose sodium was used a vehicle and Mitomycin C was used as a positive control substance. 30 mice (6 mice /group) were dosed daily at an interval of 24 hrs for 2 days. Bone marrow cells were examined. The target tissues were treated to achieve cells, slide preparation of the cells was done and later the slide was used for electrophoresis. Frequencies of micro nucleated polychromatic erythrocytes (MNPCEs) in treatment and positive control groups were compared with those in the negative control group using conditional binomial tests.In micronucleus tests, no significant differences in MNPCEs were found between test chemical treatment groups and the negative control group. The frequency of PCEs, which offers an index of the influence of the test substance on bone marrow cells, did not differ between any of the treatment groups and the negative control group. The frequency of MNPCEs in the positive control group was increased (p ≤ 0.01) in comparison with that in the negative control group. No deaths, clinical signs and body weight changes were observed in control and treatment groups. Thus the test chemical was considered to be not mutagenic in nature.

In another Mammalian Erythrocyte Micronucleus study, the mutagenic nature of test chemical was evaluated in male FVB mouse. Phosphate buffer saline was used as vehicle and monomeric acryl amide was used a positive control substance. The test chemical was administered intraperitoneally at a dose range of 0, 100, 200, 400, 600, 800, 1000, 1500 and 2000 mg/kg bw. A total of 28 mice were used in the experiment [3 mice/ dose group and 3 mice for positive control group; Group 7 (1000 mg/kg bw) consisted of 4 mice]. Liver, Kidney and brain tissue were the target organs.The test animals were sacrificed at 46 h after treatment and blood samples were collected and the frequency of micronucleated polychromatic erythrocytes (fMNPCE) and the cell proliferation (%PCE) was determined The two-tailed, Student t-test was used for a statistical evaluation of the result of the positive control, acrylamide. The linear regression line for the fMNPCE values over the entire dose interval was calculated. The analyses did not show any significant difference in the %PCE or in the fMNPCE. Consequently, under the testing circumstances one can conclude that the test chemical is not genotoxic.

Based on the observations made, the frequency of MNPCEs were comparable to control groups. Hence it is not likely to classify as a gene mutant in vivo as per the criteria mentioned in CLP regulation.

Transgenic Rodent Somatic and Germ Cell Gene Mutation Assay:

In Transgenic Rodent Somatic and Germ Cell Gene Mutation Assay, the mutagenic nature of test chemical was evaluated in male CD2-LacZ80/HazfBR mice. The test chemical was administered orally via gavage daily once for 4 weeks at a dose concentration of 250, 500 and 1000 mg/kg bw. 7, 12-dimethylbenz[a]anthracene (DMBA) wa used as a positive control. In total 28 mice were used in the experiment (6 mice/dose, 6 mice for negative control and 4 mice for positive control group).Liver and glandular stomach tissue was the target organs for examination.Mice were sacrificed by CO2 asphyxiation 3 days after the last treatment. Liver and stomach tissues were collected, stomach tissues were divided into forestomach and glandular stomach, and genomic DNA was extracted from liver and glandular stomach samples.Differences in MFs were analyzed for significance by the Conditional Binomial test (upper tailed significance level of 0.05). No deaths and no clinical signs of toxicity were observed in treatment groups. Body weight gains during treatment were the same in all treatment groups. MFs of cII genes in liver and glandular stomach tissues from test chemical-treated animals were not significantly higher than those in respective negative control animals. Thus, according to the summarized study, the test chemical was considered to be not mutagenic in nature.

In vivo Alkaline comet assay:

Data available for the test chemical was reviewed to determine the mutagenic nature of test chemical; the studies are as mentioned below:

In an in vivo Alkaline comet assay test, the mutagenic nature of test chemical was evaluated in maleCD2F1/Crlj (CDF1), SPF/VAF mouse. The mice were dosed orally by plastic syringe and stomach tube at a concentration of 500, 1000 and 2000 mg/kg bw for two days. 5 mice per dose per group along with positive and negative control groups were used. Liver lobe and surface epithelia from the glandular stomach tissues were the target tissues. The target tissues were treated to achieve cells, slide preparation of the cells was done and later the slide was used for electrophoresis. In Statistical analysis, mean percentages of tail DNA were compared between each treatment group and the negative control group using Dunnett's multiple comparison test (1-tailed), and differences were considered significant when p < 0.05 and p < 0.01. No deaths and no clinical signs of toxicity were observed in any of the treatment groups. No significant differences in numbers of hedgehogs or DNA damages were observed in either organ between test chemical treated animals and negative control animals. The positive control (EMS) group showed significant increases (p ≤0.05) in percentages of DNA in Comet tails from both organs. Thus, the test chemical was considered to be Not mutagenic In-vivo.

In vivo Mammalian Alkaline Comet Assay was performed to determine the lack of genotoxicity in-vivo of the test chemical. The experiment was performed on male Hsd:ICR (CD- 1)mouse. The test chemical was administered by oral gavage route at a dose level of 0, 25, 500 and 2000 mg/kg bw for 3 days. A total of 29 mice were used (6 mice/group and 6 mice for positive control group). Methyl methanesulfonate was used as a positive control substance.Liver, stomach and colon tissues were examined after the completion of experiment. Evaluation was done with fully validated automated scoring system Comet Assay IV.Evaluation of Comet parameters in the liver, stomach and colon of treated animals showed that the mean %Tail DNA intensity and Tail Moment values at all three dose levels were comparable to concurrent controls (mean %Tail DNA: 0.028, 1.04, 0.51, in liver, colon and stomach, respectively) and remained within the historical vehicle control range, where available (liver) (mean %Tail DNA liver: 0.024-0.025; colon: 0.59-2.17; stomach: 0.96-1.19). The % Tail DNA in samples with evidence of mechanical damage was slightly higher than the rest of the samples in the same dose group but did not impact the group means significantly. There was no statistically significant increase in DNA damage in liver, colon or stomach in animals treated orally with test chemical compared to vehicle control animals. Thus the test chemical can be considered as not mutagenic In-vivo.

Based on the observations made, there was no statistically significant increase in DNA damage in liver, colon or stomach in animals treated orally with test chemical compared to vehicle control animals. Hence it is not likely to classify as a gene mutant in vivo as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the observations made, the test chemical does not exhibit gene mutation in vitro and In-vivo. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.